Introduction

Phosphatases of the regenerating liver [PRLs; also
known as the protein tyrosine phosphatase type IVA (PTP4A) family]
were originally identified as immediate-early genes in the
regenerating liver (1). The PRL
family is a group of protein tyrosine phosphatases (PTPs) and plays
a role in the development and metastasis of various cancers,
including colorectal, prostate, breast, gastric and liver cancers,
and particularly in metastatic cancers (2,3). The PRL
family comprises three genes: PRL-1, PRL-2 and PRL-3. The
overexpression of the PRL family has been frequently reported in
various cancers, especially in metastatic cancers (4–8).
Overexpression of PRLs in normal cells has been found to promote
proliferation, migration, and invasion (4,8,9) whereas the reduction of PRLs in cancer
cells using small interfering RNA (siRNA) has been shown to inhibit
cell motility and metastatic characteristics in a mouse model
(10).

Although it is important to elucidate the role of
PRLs in cancer progression and the signaling pathways they affect,
a major challenge to the analysis of the detailed signaling
mechanism of PRLs is the lack of a physiologically relevant
substrate and knowledge of its regulation by physical interaction.
Several PRL-interacting proteins such as activating transcription
factor-7, β-subunit of geranylgeranyl transferase-II, cadherin 22,
ezrin, elongation factor 2, keratin 8, integrin-α1, PRL-1 (trimer),
PRL-3 (oligomer) and FKBP8 have been reported (1,11,16,20–27).

PRL family members have been identified to be useful
biomarkers and therapeutic targets in cancer as well as in
metastatic cancer due to the aforementioned properties (1,3,27). However, little is known about the
proteins that bind to PRL and regulate PRL function or are
regulated by PRL. Therefore, in the present study, to screen for
novel PRL-interacting proteins, yeast two-hybrid methodology was
applied using PRL-1 and PRL-3 as bait. The identification of
PRL-binding proteins may be useful in providing a novel insight
into the mechanisms of tumorigenesis and other diseases, and might
eventually lead to the development of more effective therapies.

Flag-PRL-1 and Flag-PRL-3 (12,19,28) were
digested with restriction enzymes (EcoRI/XhoI) and
cloned into the yeast expression vector pLexA (Clontech
Laboratories, Inc., Mountainview, CA, USA) to form pLexA-PRL-1 and
pLexA-PRL-3, respectively. The authenticity and correct orientation
of the cloned sequence were then confirmed by restriction digestion
and polymerase chain reaction (PCR).

Two cDNA clones encoding FKBP8 and SELPLG from
pJG4-5 (Clontech Laboratories, Inc.) were inserted into a pcHA
vector (Addgene vector database) to express their proteins in
mammalian cells. Prey genes were digested with restriction enzymes
(EcoRI/XhoI) and cloned into the mammalian expression vector pcHA.
Insertion of the prey genes were confirmed by restriction enzyme
digestion and nucleotide sequencing.

PCR

The DNA used for the PCR was obtained from bacterial
plasmid DNA (Bioneer Corporation, Daejeon, Korea). PCR was
performed with the following primer pairs: PRL-1 forward,
5′-TACACACAATCCAACCAATG-3′, and reverse,
5′-AATTAATGCTAGGGCAACAA-3′, and PRL-3 forward,
5′-TCATTGAGGACCTGAAGAAG-3′, and reverse,
5′-CTCAGCCAGTCTTCCACTAC-3′. PCR pre-mix was used for the reaction
(Bioneer Corporation). In each reaction, 20 µl final reaction
mixture contained 10 µl Premix Taq, 0.8 ml PCR forward
primer (10 mm), 0.8 ml reverse primer (10 mm), 2 µl DNA (100 ng/µl)
and dH2O. Subsequently, the reaction mixture was
incubated at 95°C for 5 min, followed by 40 cycles of 95°C for 15
sec and 60°C for 45 sec with 20 cycles. 1.5% agarose gel was used
for electrophoresis of the PCR product.

Screening of a HeLa library and
selection of proteins interacting with PRL-1 and PRL-3

Dual-luciferase assay

HeLa cells were transfected with pRGC-luc (28), along with each expression vector
(HA-SELPLG, HA-FKBP8, Flag-PRL-1 and/or Flag-PRL-3) as indicated
using Lipofectamine Plus. The cells were lysed, and the luciferase
activity was evaluated using a dual luciferase assay kit (Promega
Corporation, Madison, WI, USA). The data were normalized to the
expression levels of a cotransfected Renilla luciferase
activity reporter control.

Functional classification, pathway analysis and
protein interaction network. The 12 identified proteins were sorted
by pathway and the Gene Ontology (GO) categories using the DAVID
database. SELPLG was selected in the Biocarta pathway. For the
network of the PRL-1, PRL-3 and prey proteins, the cellular protein
interaction network was constructed based on the screened proteins
in this study and in the STRING database.

Results

Screening of interacting proteins with
PRL-1 or 3 using a yeast two-hybrid system

The PRL family plays a significant role in the
development and cancer metastasis, and shares a high degree of
sequence similarity. Notably, PRL-3 has >75% amino-acid sequence
similarity to PRL-1, with a conserved function (1,27,30).

To screen novel PRL-interacting proteins, human
PRL-1 and PRL-3 were used as bait in a yeast two-hybrid system.
Flag-PRL-1 and Flag-PRL-3 were digested with restriction enzymes
(EcoRI/XhoI) and the inserts were cloned into the
yeast expression vector pLexA (Fig.
1A). To confirm the cloning, PCR products of full length PRL-1
and PRL-3 from pLexA-PRL-1 and pLexA-PRL-3 were identified by
nucleotide electrophoresis (data not shown). In addition, the
inserts of PRL-1 and PRL-3 from pLexA-PRL-1 and pLexA-PRL-3 were
investigated by nucleotide electrophoresis following digestion with
same restriction enzymes (Fig. 1B).
Also, the sequence and the orientation of the inserts were
confirmed by sequencing analysis. Finally, the expression of the
PRL-1 bait in yeast EGY48 was confirmed by western blotting
(Fig. 1C).

In vivo binding and
colocalization

From among the 12 candidate genes interacting with
PRL-1 or PRL-3, two cDNA clones encoding for FKBP8 and SELPLG were
inserted into pcHA vector to express their proteins in mammalian
cells. Prey genes were digested with restriction enzymes
(EcoRI/XhoI) and cloned into the mammalian expression
vector pcHA. Insertion of the prey genes was confirmed by
restriction enzyme digestion and nucleotide sequencing (Fig. 1E).

To confirm their binding in a yeast-independent
interaction assay, coimmunoprecipitation experiments were
performed. HEK293T cells were co-transfected with Flag-PRL-1 and
HA-FKBP8 or HA-SELPLG constructs, and cell extracts were then
subjected to immunoprecipitation with anti-Flag antibody, followed
by immunoblotting analysis with an anti-HA antibody. HA-tagged
FKBP8 and SELPLG were detected in anti-Flag-PRL-1
immunoprecipitates (Fig. 2A).

The localization of bait proteins and prey proteins
was then examined. U2OS cells were transfected with Flag-PRL-1, and
HA-FKBP8 or HA-SELPLG. Localization of FLAG tagged-PRL-1 was
visualized with anti-FLAG primary antibody and Fluor 488-conjugated
goat antibody against mouse IgG and localization of HA-tagged preys
was visualized with anti-HA antibody and Alexa Fluor 594-conjugated
goat antibody against rabbit IgG.

In cells, PRLs are typically associated with the
plasma membrane and early endosome (1,27,30). An
important mechanism responsible for this localization is
prenylation, a post-translational lipid modification that commonly
targets proteins to membranes (3,27,30).
Fig. 2B and Table II show that PRL-1 localization is
observed in the endosome, early endosome, endoplasmic reticulum,
spindle, cytoskeleton, plasma membrane, microtubule cytoskeleton
and intracellular non-membrane-bounded organelle. SELPLG is visible
in the membrane fraction, insoluble fraction, plasma membrane, and
is integral to the plasma membrane while FKBP38 is observed in the
mitochondrial envelope, endoplasmic reticulum membrane, plasma
membrane, endomembrane system and nuclear envelope-endoplasmic
reticulum network (Fig. 2B and
Table II). The expression of SELPLG
and FKBP38 appears to be partially colocalized with PRL-1. In the
presence of preys, changes in the localization of PRL-1 were not
observed, suggesting that the expression of these preys does not
affect the prenylation and localization of PRL-1.

Table II.

Analysis of the cellular components
associated with the identified proteins, based on the cellular
components gene ontology categories of DAVID.

Table II.

Analysis of the cellular components
associated with the identified proteins, based on the cellular
components gene ontology categories of DAVID.

SELPLG and FKBP8 inhibit the functions
of PRL-1 and PRL-3

Having verified the binding of FKBP8 and SELPLG with
PRL-3 protein, the next important question is whether FKBP8 and
SELPLG affect the functions of PRL-1 and PRL-3 in cells. The roles
of PRL-1 and PRL-3 are associated with the downregulation of p21
transcription as well as the activity of p53 (28). Therefore, the effects of two prey
proteins on the downregulation of p53 reporter activities mediated
by PRL-1 and PRL-3 were investigated. HeLa cells were transfected
with each prey protein and/or Flag-PRL-1 (or Myc-PRL-3) and
p53-luciferase reporter (pRGC-luc) (Fig.
3). When p53-luc was transfected with PRL-1 or PRL-3,
inhibition of luciferase activity was observed (Fig. 3A), as shown previously (28). However, SELPLG and FKBP8 markedly
attenuated the PLR-1-mediated p53-luc inhibition (Fig. 3A). Also, similar results were
observed when SELPLG and FKBP8 were introduced with PRL-3 (Fig. 3B). These findings reveal that SELPLG
and FKBP8 inhibit the ability of PRL-1 and PRL-3 to reduce p53
reporter activity and imply that SELPLG and FKBP8 inhibit the
cellular functions of PRL-1 and PRL-3.

A PRL-1 and PRL-3-prey proteins interaction network
was constructed using the STRING database (Fig. 4). SDC4, PLIN3, SYNE2, TPD52L2, EMD
and FKBP8 were indicated to by the most closely-related and
specific node proteins associated with PRL-3, whereas SELPLG, GBP1,
RABAC1 and NDUFB8 were the most remarkable node proteins associated
with PRL-1. TMUB2 and LRP10 did not show any indirect interactions
with PRL-1 or PRL-3 (Fig. 4). These
notable node proteins appear to be particularly important in the
regulation and organization of PRL-1 and PRL-3 in the prey proteins
interaction network.

Discussion

The PRL family comprises a group of PTPs that play
an important role in the development and metastasis of various
types of cancer (12). The family
members, which include PRL-1, PRL-2 and PRL-3, share a high degree
of sequence similarity and show similar functional characteristics.
It has been reported that several signaling pathways involved in
cell growth and cancer development are affected (regulated by) PRLs
(3,4). However, the mechanisms by which PRLs
regulate signaling or interact with direct binding partners to
mediate their effects remains to be clearly elucidated.

In the present study, 12 proteins interacting with
PRL-1 or PRL-3 were identified using a yeast two-hybrid system. GO
biological process data indicated that these proteins are mostly
associated with nuclear envelope organization, endomembrane
organization and nucleus organization (Table IV). Cellular components data suggest
that they are located at membrane parts, integral to membrane,
intrinsic to membrane, envelope, nuclear membrane, contractile
fiber part, myofibril, organelle membrane and nuclear envelope
(Table II). Molecular functions of
6 genes were classified as protein binding (data not shown). They
were also found to be involved in various signaling pathways such
as oxidative phosphorylation, Alzheimer's disease, hypertrophic
cardiomyopathy, ECM-receptor interaction and cell adhesion
molecules in KEGG pathways (Table
III).

FKBP8 is a member of the FKBP family of proteins,
and is widely expressed in cancer cell lines (31,32). In
cancer, FKBP8 has potential antitumor effects via the regulation of
anti-invasive syndecan 1, proinvasive matrix metalloproteinase 9
(33,34), mechanistic target of rapamycin,
Rheb-GTP (35) and PRL-3 (28). Results of our previous study showed
that FKBP8 binds to PRL-3, and suppresses PRL-3-mediated p53
activity and cell proliferation (28). The present study also provided
evidence that FKBP8 binds to PRL-1, and suppresses the function of
PRL-1, in addition to that of PRL-3.

SELPLG is a glycoprotein that acts as a
counter-receptor for the cell adhesion molecules P-, E- and
L-selectin expressed on myeloid cells and T lymphocytes (36). In leukocyte trafficking during
inflammation, SELPLG tethers leukocytes to activating platelets or
selectin-expressing endothelia. SELPLG requires post-translational
modification by tyrosine sulfation and addition of the
sialyl-Lewis-x tetrasaccharide for its high-affinity binding
activity. Aberrant expression of and polymorphisms in the SELPLG
gene are associated with defects in the innate and adaptive immune
response.

In the present study, 12 potential PRL-1/3 binding
proteins were identified, including 11 novel binding partners and a
known binding partner, FKBP8. SELPLG and FKBP8 proteins were shown
to directly bind to PRL-1 and inhibit the downregulation of p53
reporter activities mediated by PRL-3 and PRL-1. These results
demonstrate that SELPLG and FKBP8 may be regulators of the
oncogenic proteins PRL-1 and PRL-3 and can have a marked impact on
cell proliferation.

It is possible that the 12 PRL-binding proteins
positively or negatively regulate PRL function (FKBP8 and SELPLG)
or may be regulated by PRLs. In regard to this hypothesis, further
studies are underway to reveal those mechanisms.

In conclusion, multiple PRLs binding proteins were
screened using a yeast two-hybrid system. The identified proteins
are associated with diseases including Alzheimer's disease,
Parkinson's disease, Huntington's disease, hypertrophic
cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and
dilated cardiomyopathy, suggesting that the PRL family may be
involved in diverse diseases as well as cancer. Furthermore, these
findings may provide valuable information for better understanding
the interactions between the PRL family and target proteins, and
revealing new biological functions of PRLs.

Acknowledgements

This study was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean Government
(2013-R1A1A1007596).